Biomolecule analysis kit and biomolecule analysis method

ABSTRACT

A biomolecule analysis kit includes a reaction container configured to perform an enzymatic reaction, the reaction container including a base portion which has a container-shaped portion and a low-adsorption structural portion which is provided on at least the inner surface of the container-shaped portion, the low-adsorption structural portion having an adsorption rate lower than the base portion at which at least one of a sample which becomes a target of analysis in the enzymatic reaction and a reagent for the enzymatic reaction is adsorbed thereonto, wherein a signal resulting from the enzymatic reaction is configured to be detected when the enzymatic reaction is performed in the reaction container.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application based on a PCT PatentApplication No. PCT/JP2015/052803, filed Feb. 2, 2015, whose priority isclaimed on Japanese Patent Application No. 2014-017942, filed Jan. 31,2014, the entire content of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a biomolecule analysis kit and abiomolecule analysis method.

Description of the Related Art

It is known that a disease or a physical predisposition may be diagnosedthrough biomolecular analysis. For example, a physical predispositionmay be diagnosed through Single Nucleotide Polymorphism (SNP) analysis,whether or not an anticancer drug will be administered may be determinedthrough somatic mutation analysis, and infection control measures may bedesigned based on the analysis of the protein or DNA of viruses.

In recent years, through human genome analysis performed worldwide,sequences of about 3.1 billion base pairs of the human genome have beenrevealed, and it has become evident that the number of human genes isabout 30,000 to 40,000. The base sequence of human beings varies betweenindividuals, and a variation in the base sequence that exists more than1% of a specific population is called a genetic polymorphism. Amonggenetic polymorphisms, SNP is considered to be associated with variousdiseases.

For example, genetic diseases of human beings are considered to becaused by SNP in a single gene. Furthermore, SNP in a plurality of genesis considered to affect the life style-related diseases, cancer, and thelike. Therefore, it is considered that the analysis of SNP is extremelyeffective in the development of pharmaceutical products such as in thesearch for potential drug targets or the prediction of side effects.Accordingly, SNP analysis is being pushed forward as a large globalproject.

As one factor causing a degree of efficacy or a side effect of a drug tovary between individuals, a difference of an enzyme group involved inthe drug metabolism of individuals may be exemplified. Recently, it hasbecome evident that such a difference may also result from a slightgenetic difference such as an SNP.

In recent years, a method of selecting an optimal drug by prior geneticanalysis of a patient and administering it to a patient has beenconsidered. In addition, the significance of genetic diagnosis hasrapidly become highly appreciated, not only for single-gene disordersbut also for multifactorial disorders. The efficacy of drugs targetingpathogenic bacteria or viruses varies between individuals in some caseseven in the same species, and this variation results from minute geneticdifferences between individuals in many cases. It is expected that thetest targets will greatly increase in the future in the geneticdiagnosis of pathogenic bacteria or viruses as extrinsic factorsdescribed above.

As described above, in medical treatment of the post-genomic era, theability to analyze for a minute genetic difference between human beingsor pathogenic microorganisms, particularly, the ability to analyze foran SNP is important, and it is expected that this importance willincrease in the future.

In the related art, various methods for analyzing for a minutedifference in a base sequence, particularly, for analyzing for an SNPhave been examined (see Landegren, Laboratory protocols for mutationdetection, Oxford university press, 1996, and Ahmadian et al.,Biotechniques, Vol. 32, pp 1122-1137, 2002). In order to performanalysis at a practical level, such methods are required to be excellentin all of aspects such as low cost, simplicity of the method, a shortsignal detection time, and accuracy of the detection result. However, amethod satisfying all of the above requirements has not become known sofar.

In a case where an SNP is analyzed for, generally, the sample containsonly a small amount of target gene fragment. In this case, the targetgene needs to be amplified in advance by a certain method. As a rapidgene amplification method with high reproducibility, a Polymerase ChainReaction (PCR) method is well known in the related art.

Generally, in order to detect a difference of a single base of a targetgene, it is necessary to perform a two-stage operation consisting of agene amplification stage using the PCR method or the like and a stage ofinvestigating a difference of a single base of the amplified gene.However, because this method requiring a two-stage operation includes aplurality of operations, the process thereof is complicated.Furthermore, in a PCR method, the temperature needs to be increased ordecreased. Therefore, the size of a device is increased, aheat-resistant reaction container is required, and measures forpreventing evaporation of the reaction solution need to be provided.

As an SNP detection method not requiring a two-stage reaction, there isan INVADER method. Because an INVADER method does not require PCRamplification and can cause an isothermal reaction, the size of a devicecan be reduced. However, since the INVADER method does not include agene amplification step, a signal is slowly amplified, and the reactionneeds to be performed for several hours to perform detection anddetermination. The INVADER method is a detection method using anenzymatic reaction. Particularly, as a method of shortening the time ittakes for the signal concentration to become saturated during signalamplification using an enzyme, a method of performing a reaction in amicrospace may be considered.

If the INVADER reaction is performed in a microspace, the number ofmolecules as a target of analysis contained in a single well can becomeequal to or less than 1, and the molecule as a target of analysis isseemingly in a concentrated state. Accordingly, the time taken for thesignal to become saturated can be shortened. In addition, because thenumber of molecules to be detected that is put into a single well isequal to or less than 1, by counting the number of wells from whichsignals are obtained, the concentration of the molecules to be detectedcan be accurately ascertained.

For example, Japanese Unexamined Patent Application, First PublicationNo. 2004-309405 discloses that a genetic test can be performed byperforming an enzymatic reaction in a microspace having a volume ofequal to or less than 1 pl.

If PCR is used for SNP analysis, detection can be performed in a shorttime, but the constitution of the device or the procedure becomescomplicated. Furthermore, in the isothermal reaction not using PCR, ittakes a long time until the SNP analysis is completed, and thereactivity is low. Therefore, those methods of the related art areimpractical.

SUMMARY

The present invention has been made to solve the above problems, and anobject thereof is to provide a biomolecule analysis kit and abiomolecule analysis method which make it possible to rapidly andquantitatively analyze biomolecules and to improve reactivity.

A biomolecule analysis kit according to a first aspect of the presentinvention includes a reaction container configured to perform anenzymatic reaction, the reaction container including a base portionwhich has a container-shaped portion and a low-adsorption structuralportion which is provided on at least the inner surface of thecontainer-shaped portion, the low-adsorption structural portion havingan adsorption rate lower than the base portion at which at least one ofa sample which becomes a target of analysis in the enzymatic reactionand a reagent for the enzymatic reaction is adsorbed thereonto, whereina signal resulting from the enzymatic reaction is configured to bedetected when the enzymatic reaction is performed in the reactioncontainer.

The low-adsorption structural portion may have a lower adsorption ratethan that of the base portion at which the sample is absorbed thereonto,and a background in the signal detection may be lower than a case inwhich the base portion is exposed in the reaction container.

The low-adsorption structural portion may have a lower adsorption ratethan that of the base portion at which the sample is absorbed thereonto,and a signal intensity in the signal detection may be higher than a casein which the base portion is exposed in the reaction container.

The reaction container may further have a modified portion formed bymodifying the surface of the base portion such that the adsorption ratein the base portion within the inner surface of the container-shapedportions becomes lower than the adsorption rate in the base portion, andthe container-shaped portions may have a bottomed cylindrical shapehaving an approximately circular opening portion having a diameter ofequal to or less than 5 μm.

The reaction container may further have a low-adsorption substance layerlaminated on the base portion such that the adsorption rate within theinner surface of the container-shaped portions becomes lower than theadsorption rate in the base portion, and the container-shaped portionsmay have a bottomed cylindrical shape having an approximately circularopening portion having a diameter of equal to or less than 5 μm.

A biomolecule analysis kit according to a second aspect of the presentinvention includes a reaction container configured to perform anenzymatic reaction, the reaction container including a container-shapedportion configured so that a sample that becomes a target of analysis issupplied to the container-shaped portion and a base portion in which thecontainer-shaped portion is formed, and a reagent configured to besupplied to the reaction container and is used for the enzymaticreaction, wherein the reagent contains an adsorption inhibitor forreducing an adsorption rate of at least one of the sample and thereagent with respect to the base portion, and a signal resulting fromthe enzymatic reaction is configured to be detected when the enzymaticreaction is performed in the reaction container.

The enzymatic reaction may be an isothermal reaction.

The sample which becomes a target of analysis may include any one ofDNA, RNA, miRNA, mRNA, and a protein, and a target substance of analysismay be any one of DNA, RNA, miRNA, mRNA, and a protein.

The target substance of analysis may be a nucleic acid, and theenzymatic reaction may be an INVADER reaction.

The reagent may generate a signal by any one of fluorescence, lightemission, pH, light absorption, and electric potential.

The adsorption inhibitor may be a surfactant.

The surfactant may be a nonionic surfactant.

The nonionic surfactant may be TWEEN 20.

The nonionic surfactant may be TRITON-100.

The concentration of the surfactant may be equal to or greater than0.0005% and equal to or less than 5%.

A biomolecule analysis method according to a third aspect of the presentinvention uses the biomolecule analysis kit according to the first orsecond aspect.

A biomolecule analysis kit according to a fourth aspect of the presentinvention includes a reaction container configured to perform anenzymatic reaction, the reaction container including a container-shapedportion configured so that a sample is supplied through a flow channelto the container-shaped portion and a base portion in which thecontainer-shaped portion is formed, and a reagent configured to besupplied to the reaction container and used for the enzymatic reaction,wherein the reagent contains a surfactant for reducing the surfacetension of the reagent, and fluorescence or a colorimetric signalresulting from the enzymatic reaction is configured to be detected whenthe enzymatic reaction is performed in the reaction container.

A biomolecule analysis method according to a fifth aspect of the presentinvention includes feeding a reagent into a flow channel in a reactioncontainer which has the flow channel and a plurality of wells such thatthe plurality of wells is filled with the reagent; and feeding the oilsealing solution into the flow channel and sealing the reagent into theplurality of wells with an oil sealing solution, thereby forming theplurality of wells into a plurality of independent reaction containersfor nucleic acid detection, wherein any one of the reagent and the oilsealing solution contains a surfactant.

The biomolecule analysis method according to the fifth aspect of thepresent invention may further include a step of filling the plurality ofcontainer-shaped portions with a wash buffer through the flow channelbefore filling the plurality of wells with the reagent.

A biomolecule analysis method according to a sixth aspect of the presentinvention is a biomolecule analysis method using the biomoleculeanalysis kit according to the first, second, or fourth aspect, in whichafter the wash buffer is supplied to the container-shaped portion, thereagent is supplied to the container-shaped portion.

According to the above aspects of the present invention, a biomoleculeanalysis kit and a biomolecule analysis method which makes it possibleto rapidly and quantitatively analyze biomolecules and to improvereactivity can be provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sectional view of a biomolecule analysis kit applied to abiomolecule analysis method according to a first embodiment of thepresent invention.

FIG. 2 is a flowchart of the biomolecule analysis method according tothe first embodiment of the present invention.

FIG. 3 shows fluorescence images showing the results of a fluorescenceamount measurement test in a first example of the present invention.

FIG. 4 is a graph showing the results of a fluorescence intensitymeasurement test in the first example of the present invention.

FIG. 5 is a table showing the results of a reaction time measurementtest in the first example of the present invention.

FIG. 6 is a sectional view of a biomolecule analysis kit applied to abiomolecule analysis method according to a second embodiment of thepresent invention.

FIG. 7 is a sectional view of the biomolecule analysis kit applied tothe biomolecule analysis method according to the second embodiment ofthe present invention.

FIG. 8 is a sectional view of the biomolecule analysis kit applied tothe biomolecule analysis method according to the second embodiment ofthe present invention.

FIG. 9 is a flowchart showing the biomolecule analysis method accordingto the second embodiment of the present invention.

FIG. 10 is an electron micrograph showing wells in a second example ofthe present invention.

FIG. 11A is a fluorescence image showing the result of a fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0% anda heating time of 10 minutes.

FIG. 11B is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.0005%and a heating time of 10 minutes.

FIG. 11C is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.001%and a heating time of 10 minutes.

FIG. 11D is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.005%and a heating time of 10 minutes.

FIG. 11E is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.05%and a heating time of 10 minutes.

FIG. 11F is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.5%and a heating time of 10 minutes.

FIG. 11G is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 5% anda heating time of 10 minutes.

FIG. 11H is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.0005%and a heating time of 20 minutes.

FIG. 11I is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.001%and a heating time of 20 minutes.

FIG. 11J is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.005%and a heating time of 20 minutes.

FIG. 11K is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.05%and a heating time of 20 minutes.

FIG. 11L is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 0.5%and a heating time of 20 minutes.

FIG. 11M is a fluorescence image showing the result of the fluorescenceamount measurement test performed in the second example of the presentinvention under the conditions of a concentration of TWEEN 20 of 5% anda heating time of 20 minutes.

FIG. 12 is a graph showing the results of a fluorescence intensitymeasurement test in the second example of the present invention.

FIG. 13 is a view showing the operation and effect of the biomoleculeanalysis method according to the second embodiment of the presentinvention.

FIG. 14A is a fluorescence image showing the result of a fluorescenceamount measurement test on a sample 1 in a third example of the presentinvention.

FIG. 14B is a fluorescence image showing the result of a fluorescenceamount measurement test on a sample 2 in the third example of thepresent invention.

FIG. 14C is a fluorescence image showing the result of a fluorescenceamount measurement test on a sample 3 in the third example of thepresent invention.

FIG. 14D is a fluorescence image showing the result of a fluorescenceamount measurement test on a sample 4 in the third example of thepresent invention.

FIG. 14E is a fluorescence image showing the result of a fluorescenceamount measurement test on a sample 5 in the third example of thepresent invention.

FIG. 14F is a fluorescence image showing the result of a fluorescenceamount measurement test on a sample 6 in the third example of thepresent invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS First Embodiment

Hereinafter, a biomolecule analysis kit and a biomolecule analysismethod according to a first embodiment of the present invention will bedescribed with reference to FIGS. 1 and 2.

FIG. 1 is a sectional view of the biomolecule analysis kit to which thebiomolecule analysis method according to the present embodiment isapplicable. In the biomolecule analysis kit according to the presentembodiment, as a biomolecule to be analyzed, any of DNA, RNA, miRNA,mRNA (hereinafter, referred to as RNAs in some cases), and a protein isselected.

As shown in FIG. 1, a biomolecule analysis kit 100 includes a soft flatplate 12 and a glass substrate 14 that constitute a reaction container10 and a cover glass 13 that can seal off the reaction container 10.

The reaction container 10 has a base portion 2 which is formed to havemicrospaces 11 (container-shaped portions) each having a bottomedcylindrical shape with one open end and low-adsorption structuralportions 3 which are provided on the surface of the base portion 2.

By preparing the microspaces 11 in the soft flat plate 12 formed ofpolydimethylsiloxane (PDMS) by means of imprinting, the reactioncontainer 10 is formed.

The microspace 11 constituting the reaction container 10 is a spacehaving a bottomed cylindrical shape with an opening portion at one end.The microspace 11 has a diameter L1 of 5 μm and a depth L2 of 5 μm, forexample. For instance, the microspace 11 has a volume of about 100femtoliters (fl). In the reaction container 10, an array of a pluralityof microspaces 11 is formed. That is, the microspaces 11 are arrayed inthe reaction container 10.

For example, on the 5 mm×5 mm rectangular surface of the soft flat plate12, the microspaces 11 are arrayed in the form of a lattice along eachside of the surface. The size of a gap between the microspaces 11 is setaccording to the resolution by which a signal can be independentlydetected in each of the microspaces 11.

The volume of each microspace 11 may be appropriately set. However, thesmaller the volume of the microspace 11, shorter the reaction time untila signal becomes detectable. For example, the volume of the microspace11 is equal to or less than 100 picoliters (pl).

Specifically, in a case where the aim is to shorten the time taken forgenerating a sufficient signal by saturating the signal, the volume ofthe microspace 11 is set based on a liquid amount in which the number ofbiomolecules as a target of analysis becomes equal to or less than 1 perwell.

The soft flat plate 12 is formed on the glass substrate 14, for example.The thickness of the glass substrate 14 is appropriately set inconsideration of a point that the glass substrate 14 needs to have asufficient strength in a process of forming the plurality of microspaces11 by means of imprinting by using the soft flat plate 12 as a material.

In the present embodiment, the low-adsorption structural portions 3 havethe following constitution, for example.

Constitutional Example 1

In each low-adsorption structural portion 3, a region positioned on theinner surface of the microspace 11 of the reaction container 10 withinthe surface of the base portion 2 is hydrophobic. For instance, eachlow-adsorption structural portion 3 has a modified portion 4 formed bymodifying the surface of the base portion 2 to be hydrophobic.

Constitutional Example 2

Each low-adsorption structural portion 3 has a low-adsorption substancelayer 4A in a region positioned on the inner surface of the reactioncontainer 10 within the surface of the base portion 2. Thelow-adsorption substance layer 4A is formed of a material to which asample as a target of analysis using the biomolecule analysis kit 100 ofthe present embodiment or a reagent for the analysis exhibits a lowadsorption rate. For example, the low-adsorption substance layer 4A is ahydrophobic coat.

Examples of the low-adsorption substance layer 4A also include a polymercoat having a molecular structure that does not allow the permeation ofa fluorescent substance. The polymer coat preferably has a molecularstructure denser than that of PDMS described above. By inhibiting thepermeation of a fluorescent substance, the polymer coat exerts an effectof preventing the decrease of signal intensity. For substances otherthan PDMS, based on the molecular structure of the substance thatbecomes the material of the base portion 2, a polymer coat having amolecular structure that can prevent the permeation of a reagent may beselected. Such a polymer coat inhibits a decrease of signal intensity.

The polymer coat in the low-adsorption structural portions 3 is notlimited to the coat inhibiting the permeation of a fluorescentsubstance, and a coat that inhibits the permeation of a substanceinvolved in an enzymatic reaction may be appropriately selectedaccording to the reagent to be used.

Next, the composition of the reagent which can be suitably used in thebiomolecule analysis kit 100 according to the present embodiment will bedescribed.

In the present embodiment, each reagent contains an adsorptioninhibitor, and accordingly, the constituents of the reagent can beprevented from being adsorbed onto the inner surface of the reactioncontainer 10 of the biomolecule analysis kit 100.

For example, the adsorption inhibitor has a composition containing atleast one kind of component among a surfactant, phospholipid, and otherpolymer compounds, and any materials may be used by being mixedtogether. Examples of the surfactant include a nonionic surfactant.Examples of the nonionic surfactant include TWEEN, glycerol,TRITON-X100, and the like. Examples of the polymer compounds includepolyethylene glycol (PEG), DNA, and a protein.

Examples of the adsorption inhibitor as a mixture of two or more kindsof material include an adsorption inhibitor obtained by mixingphospholipid with PEG

In a case where a nonionic surfactant is used as the surfactant, theconcentration of the nonionic surfactant contained in the reagent ispreferably equal to or less than 5%. In a case where TWEEN 20 is used,the concentration of TWEEN 20 contained in the reagent is preferablywithin a range of equal to or greater than 0.0005% and equal to or lessthan 5%, and more preferably within a range of equal to or greater than0.001% and equal to or less than 0.5%. If the concentration of TWEEN 20is equal to or greater than 0.0005%, the reactions caused in theplurality of microspaces 11 can be independently detected, and thefluorescence from the microspaces 11 can be accurately measured. If theconcentration of TWEEN 20 is equal to or less than 5%, a sufficientenzymatic reaction is obtained.

The adsorption inhibitor described above may be a substance adsorbedonto the inner surface of the microspace 11 in the reaction container10. By supplying the adsorption inhibitor-containing reagent into thereaction container 10, the adsorption inhibitor is adsorbed onto theinner surface of the reaction container 10. As a result, compared to acase where the reagent does not contain the adsorption inhibitor, theenzyme used for the enzymatic reaction, the nucleic acid or protein thatbecomes a target of analysis, the labeling substance used for signaldetection, and the like are hardly adsorbed onto the inner surface ofthe reaction container 10.

In a case where oil is put into the microspaces 11, the adsorptioninhibitor may be added to the oil to be used.

It is preferable that the adsorption inhibitor is contained in at leastany one of the reagents coming into contact with the inside of thereaction container 10 during the period between a time before at leastone of the enzyme used for the enzymatic reaction, the nucleic acid orprotein that becomes a target of analysis, the labeling substance usedfor signal detection, and the like is initially supplied into thereaction container 10 and a time when the signal detection is ended. Forexample, the adsorption inhibitor may be mixed with a solvent such as abuffer solution for diluting the reagent at a predeterminedconcentration.

The adsorption inhibitor may be contained in all of the reagents cominginto contact with the inside of the reaction container 10 during theperiod between a time before at least one of the enzyme used for theenzymatic reaction, the nucleic acid or protein that becomes a target ofanalysis, the labeling substance used for signal detection, and the likeis initially supplied into the reaction container 10 and a time when thesignal detection is ended.

The adsorption inhibitor is preferably a substance that does not hinderan enzymatic reaction or a signal amplification reaction.

Next, a biomolecule analysis method using the biomolecule analysis kit100 according to the present embodiment will be described. FIG. 2 is aflowchart showing the biomolecule analysis method according to thepresent embodiment.

First, a reagent containing a substance (DNA in the present embodiment,for example) that becomes a target of analysis is added dropwise to themicrospaces 11 of the reaction container 10 (Step S101 shown in FIG. 2).Specifically, the reagent added dropwise in the present embodimentcontains an INVADER reaction reagent (1 μM allele probe, 0.4 μM INVADERoligo, 1 μM FAM-labeled arm, 20 mM MOPS pH 7.5, 15 mM NaCl, 6.25 mMMgCl₂, and 50 U/μL cleavase) and DNA.

The liquid amount of the reagent added dropwise to the microspaces 11 ofthe reaction container 10 may be appropriately set according to thenumber of microspaces 11. Furthermore, the liquid amount andconcentration of the reagent added dropwise to the microspaces 11 of thereaction container 10 are adjusted such that approximately single DNAmolecule is put into each of the microspaces 11. For example, in thepresent embodiment, the liquid amount of the reagent added dropwise tothe microspaces 11 of the reaction container 10 is 0.5 μL in total, and0.5 μL of the liquid is distributed into the plurality of microspaces11.

Then, the microspaces 11 of the reaction container 10 are covered withthe cover glass 13 (Step S102 shown in FIG. 2). As a result, each of themicrospaces 11 becomes an independent reaction chamber filled with theINVADER reaction reagent and DNA. Thereafter, the reaction container 10in which the microspaces 11 are filled with the INVADER reaction reagentand DNA is incubated in an oven at 62° C., for example (Step S103 shownin FIG. 2). Through the incubation, signal amplification performed in anisothermal INVADER reaction suitably proceeds.

Subsequently, the reaction container 10 in which each of the microspaces11 is filled with the INVADER reaction reagent and DNA is taken outafter a preset time, and the number of wells emitting fluorescence andthe fluorescence amount thereof are measured (Step S104 shown in FIG.2).

In the present embodiment, a detection system may also be adopted whichdetects, in addition to fluorescence, emission of visible light, colordevelopment, a change in pH, a change in electric potential, or the likeas a signal. In addition, the constitution of the present embodiment canbe adopted for analyzing proteins.

Second Embodiment

Hereinafter, a biomolecule analysis kit and a biomolecule analysismethod according to a second embodiment of the present invention will bedescribed with reference to FIG. 6. A biomolecule analysis kit 100Aaccording to the present embodiment includes an array device 20 fornucleic acid quantification, reagent, and an oil sealing solution.

FIG. 6 is a sectional view of the array device 20 for nucleic acidquantification according to the present embodiment. In the biomoleculeanalysis kit according to the present embodiment, as a biomolecule to beanalyzed, any of DNA, RNA, miRNA, mRNA (hereinafter, referred to as RNAsin some cases), and a protein is selected.

As shown in FIG. 6, the array device 20 for nucleic acid quantificationincludes a reaction container 30, a cover portion 27, an inlet portion(not shown in the drawing), and an outlet portion (not shown in thedrawing). The reaction container 30 includes a base portion 23 and aflow channel 31. In the base portion 23, a plurality of wells(container-shaped portions) 26, a substrate 24, and a micropore arraylayer 25 are formed.

The micropore array may be formed directly in the substrate 24.Alternatively, a member in which the micropore array is formed may befixed to the substrate 24 by means of adhesion, welding, or the like.

The substrate 24 is a plate-like member formed of a material that issubstantially transparent. The material of the substrate 24 is a resinor glass, for example. Specifically, the substrate 24 may be formed ofpolystyrene or polypropylene. The substrate 24 should have such astiffness that the substrate is not broken at the time of handling dueto a device for transporting the array device 20 for nucleic acidquantification or a manual operation of an operator.

The micropore array layer 25 is a layer in which a plurality of throughholes 25 a arranged in a line. The thickness of the micropore arraylayer 25 is 3 μm, and there is a clearance of 100 μm between themicropore array layer 25 and the cover portion 27. Each of the throughholes 25 a is a bottomed cylindrical space having an opening portion atone end, and has a diameter of 5 μm (the through hole 25 a has acylindrical shape 3 μm long in the centerline direction). For example,the volume of the through hole 25 a is about 60 femtoliters (fl).

The volume of each through hole 25 a may be appropriately set. However,the smaller the volume of the through hole 25 a, the further thereaction time taken until a signal becomes detectable can be shortened.

For example, the volume of each through hole 25 a is equal to or lessthan 100 picoliters.

In the present embodiment, a detection system may also be adopted whichdetects, in addition to fluorescence, emission of visible light, colordevelopment, a change in pH, a change in electric potential, or the likeas a signal. In addition, the constitution of the present embodiment canbe adopted for analyzing proteins.

The distance (pitch) between the center lines of the through holes 25 ashould be longer than the diameter of each of the through holes 25 a.

The size of the interval (gap) between the through holes 25 a is setaccording to the resolution by which a signal can be independentlydetected in each of the through holes 25 a.

The through holes 25 a are arranged to form a triangular lattice shapewith respect to the micropore array layer 25.

The way the through holes 25 a are arranged is not particularly limited.In the base portion 23, bottomed cylindrical micro-wells 26(container-shaped portions) in which the substrate 24 becomes a bottomsurface portion 26 a are formed by the through holes 25 a formed in themicropore array layer 25 and a surface 24 a of the substrate 24.

Specifically, in a case where the aim is to shorten the time taken forgenerating a sufficient signal by saturating the signal, the volume ofeach well 26 is set based on a liquid amount in which the number ofbiomolecules as a target of analysis becomes equal to or less than 1 perwell.

The material of the micropore array layer 25 may be a resin, glass, andthe like, and may be the same as or different from the material of thesubstrate 24. Furthermore, the micropore array layer 25 and thesubstrate 24 may be integrated by the same material. In addition, themicropore array layer 25 and the substrate 24 may be integrally moldedwith the same material. Examples of the material of the micropore arraylayer 25 formed of a resin include a cycloolefin polymer, silicon,polypropylene, polycarbonate, polystyrene, polyethylene, polyvinylacetate, a fluorine resin, an amorphous fluorine resin, and the like.These are merely examples of the material of the micropore array layer25, and the material of the micropore array layer 25 is not limitedthereto.

The micropore array layer 25 may be colored. If the micropore arraylayer 25 is colored, in a case where optical measurement such as themeasurement of fluorescence, light emission, absorbance, and the like isperformed in the wells 26, the influence of light from other wells 26adjacent to a well 26 that becomes a measurement target can be reduced.

By performing processing such as etching, embossing, or cutting on thesolid pattern of a hydrophobic coat laminated on the substrate 24, thethrough holes 25 a are formed on the micropore array layer 25. In a casewhere the micropore array layer 25 and the substrate 24 are integrallymolded, by performing processing such as etching, embossing, or cuttingon the substrate 24, the portions corresponding to the through holes 25a of the micropore array layer 25 are formed. In this way, a patternhaving a hydrophobic portion and a hydrophilic portion can be formed onthe substrate.

The cover portion 27 is superposed on the base portion 23 such that thecover portion 27 covers opening portions of the plurality of wells 26 ina state where a gap is formed between the base portion 23 and the coverportion 27. The space between the base portion 23 and the cover portion27 becomes a flow channel 31 through which various liquids flow. In thepresent embodiment, through the space between the base portion 23 andthe cover portion 27, various liquids flow from the inlet portion towardthe outlet portion.

Next, the composition of the reagent which can be suitably used in thebiomolecule analysis kit 100A according to the present embodiment willbe described.

As shown in FIGS. 7 and 8, a detection reaction reagent 21 is a solutionwhich can be fed into the space between the base portion 23 and thecover portion 27 from the inlet portion. The detection reaction reagent21 is a reagent for causing a biochemical reaction such as an enzymaticreaction with regard to a target substance of analysis.

The biochemical reaction to a target substance of analysis is a reactionby which signal amplification may occur in the presence of a nucleicacid in a case where the target substance of analysis is DNA (nucleicacid), for example. The detection reaction reagent 21 is selectedaccording to the method which can detect a nucleic acid, for example.For instance, the reagents used in an INVADER (registered trademark)method, a LAMP (registered trademark) method, a TAQMAN (registeredtrademark) method, a fluorescent probe method, or other methods areincluded in the detection reaction reagent 21 of the present embodiment.

In the present embodiment, when the target substance of analysis is anucleic acid, the nucleic acid can be detected without performing anucleic acid amplification step as in the PCR method as in the relatedart. However, if necessary, a product obtained by amplifying the nucleicacid as a target of analysis by the PCR method or the like may be usedas a sample.

Furthermore, even when the target substance of analysis is other than anucleic acid, the present embodiment can be applied after the substanceis appropriately pre-treated as necessary such that it becomesapplicable to the present embodiment.

In the present embodiment, at least one of the reagents contains theadsorption inhibitor, and as a result, the constituents of the reagentcan be prevented from being adsorbed onto the inner surface of the wells26 of the biomolecule analysis kit 100A. All of the reagents may containthe adsorption inhibitor.

Examples of the reagents include a buffer, a detection reaction reagent,a sample (a substance as a target of analysis: DNA, RNAs, a protein, orthe like) solution, a sealing solution, and a solvent for diluting areagent or a sample.

For example, the adsorption inhibitor has a composition containing atleast one kind of component among a surfactant, phospholipid, and otherpolymer compounds, and any materials may be used by being mixedtogether. Examples of the surfactant include a nonionic surfactant.Examples of the nonionic surfactant include TWEEN, glycerol,TRITON-X100, and the like. Examples of the polymer compounds includepolyethylene glycol (PEG), DNA, and a protein.

Examples of the adsorption inhibitor as a mixture of two or more kindsof materials include an adsorption inhibitor obtained by mixingphospholipid with PEG

In a case where a nonionic surfactant is used as the surfactant, theconcentration of the nonionic surfactant contained in a reagent ispreferably equal to or less than 5%. In a case where TWEEN 20 is used,the concentration of TWEEN 20 contained in a reagent is preferablywithin a range of equal to or greater than 0.0005% and equal to or lessthan 5%, and more preferably within a range of equal to or greater than0.001% and equal to or less than 0.5%. If the concentration of TWEEN 20is equal to or greater than 0.0005%, the reactions caused in theplurality of wells 26 can be independently detected, and thefluorescence from the wells 26 can be accurately measured. If theconcentration of TWEEN 20 is equal to or less than 5%, a sufficientenzymatic reaction is obtained.

The surfactant is not limited to the nonionic surfactant, and an ionicsurfactant (an anionic, cationic, or amphoteric surfactant) may be usedas the surfactant. A mixture of ionic surfactants or a mixture of anionic surfactant and a nonionic surfactant may also be used.

Furthermore, a mixture of a surfactant and a polymer compound can alsobe used as the adsorption inhibitor.

Next, the composition of the oil sealing solution 22 which can besuitably applied to the biomolecule analysis kit 100A according to thepresent embodiment will be described.

In the present embodiment, for the purpose of preventing theconstituents of the reagent from being adsorbed onto the inner surfaceof the wells 26 of the biomolecule analysis kit 100A, the adsorptioninhibitor may be contained in the oil sealing solution 22.

The oil sealing solution 22 (see FIG. 8) is a solution which can be fedinto the space between the base portion 23 and the cover portion 27 fromthe inlet portion. The oil sealing solution 22 can be selected from thematerials which are immiscible with the sample containing the targetsubstance of analysis. As the oil sealing solution 22, mineral oil, FC40as a fluorine-based liquid, or the like can be used.

Furthermore, in the present embodiment, for the purpose of preventingthe constituents of the reagent from being adsorbed onto the innersurface of the wells 26 of the biomolecule analysis kit 100A, a washbuffer for wells may be fed into the kit before the reagent is fed intothe kit. The buffer may contain the adsorption inhibitor.

The adsorption inhibitor may be a substance adsorbed onto the innersurface of the wells 26 in the reaction container 30. By supplying theadsorption inhibitor-containing reagent into the reaction container 30,the adsorption inhibitor is adsorbed onto the inner surface of thereaction container 30. As a result, compared to a case where the reagentdoes not contain the adsorption inhibitor, the enzyme used for theenzymatic reaction, the nucleic acid or protein that becomes a target ofanalysis, the labeling substance used for signal detection, and the likeare hardly adsorbed onto the inner surface of the reaction container 30.

The adsorption inhibitor contained in the wash buffer may be a nonionicsurfactant. Examples of the nonionic surfactant include TWEEN, glycerol,TRITON-X100, and the like. Furthermore, the wash buffer may constitute aportion of the reagent.

It is preferable that the adsorption inhibitor is contained in at leastone of the reagents coming into contact with the inside of the reactioncontainer 30 during the period between a time before at least one of theenzyme used for the enzymatic reaction, the nucleic acid or protein thatbecomes a target of analysis, the labeling substance used for signaldetection, and the like is initially supplied into the reactioncontainer 30 and a time when the signal detection is ended. For example,the adsorption inhibitor may be mixed with a solvent such as a buffersolution for diluting the reagent at a predetermined concentration.

The adsorption inhibitor may be contained in all of the reagents cominginto contact with the inside of the reaction container 30 during theperiod between a time before at least one of the enzyme used for theenzymatic reaction, the nucleic acid or protein that becomes a target ofanalysis, the labeling substance used for signal detection, and the likeis initially supplied into the reaction container 10 and a time when thesignal detection is ended.

The adsorption inhibitor is preferably a substance that does not hinderan enzymatic reaction or a signal amplification reaction.

Next, a biomolecule analysis method using the biomolecule analysis kit100A according to the present embodiment will be described. FIG. 9 is aflowchart showing the biomolecule analysis method according to thepresent embodiment.

First, the inlet portion and the outlet portion not shown in the drawingare opened, and a wash buffer 33 containing the adsorption inhibitor isfed into the gap between the base portion 23 and the cover portion 27through the inlet portion by a dispensing pipette, for example (StepS201 shown in FIG. 9). The buffer 33 spreads across the gap between thebase portion 23 and the cover portion 27 so as to cover all of theplurality of wells 26 (see FIG. 6). As a result, within the surface ofthe base portion 23, low-adsorption structural portions 32 having alow-adsorption substance layer 35 is formed in a region positioned onthe inner surface of the through hole 25 a and a region 34 positionedbetween wells adjacent to each other.

The reaction container 30 may be filled in advance with the buffer 33instead of being fed with the buffer 33. In this case, the inlet portionand the outlet portion may be sealed with a film or the like such thatthe buffer 33 is sealed in the reaction container 30.

Then, a reagent containing a substance (in the present embodiment, DNAfor example) that becomes a target of analysis is fed into the gapbetween the base portion 23 and the cover portion 27 through the inletportion by a dispensing pipette, for example (Step S202 shown in FIG.9). Specifically, the reagent that fills the kit in the presentembodiment contains an INVADER reaction reagent (the detection reactionreagent 21) (1 μM allele probe, 1 μM of INVADER oligo, 1 μM FAM-labeledarm, 10 mM MOPS pH 7.5, 6.25 mM MgCl₂, and 50 U/μL cleavase, and Tween20) and DNA which is a target substance of analysis. The reagent spreadsacross the gap between the base portion 23 and the cover portion 27 soas to cover all of the plurality of wells 26 (see FIG. 7). The reagentis fed into the gap between the base portion 23 and the cover portion27, and as a result, the buffer 33 is discharged from the outletportion. At this time, if the color of the reagent is different fromthat of the buffer 33, it is easy to ascertain into which portion thereagent has been fed within the space between the base portion 23 andthe cover portion 27.

As shown in FIG. 6, in the flow channel 31 formed by the base portion 23and the cover portion 27, the plurality of wells 26 formed by thesubstrate 24 and the micropore array layer 25 is arranged. As thereagent is flowed into the wells 26, the buffer 33 that fills theplurality of wells 26 is sequentially replaced with the reagent.

However, some of the wells 26 retain the buffer 33 on the inner surfacethereof. In this case, the buffer 33 that fills the plurality of wells26 is not replaced with the reagent, and the reagent is superposed onthe buffer 33. However, because the buffer 33 and the reagent are easilyintermixed, after the reagent is superposed on the buffer 33, the solutein the reagent is diffused into the buffer 33. Therefore, in both of thewell in which the buffer is replaced with the reagent and the well inwhich the reagent is superposed on the buffer 33, substantially the samereaction occurs.

The amount of liquid that fills the wells 26 may be appropriately setaccording to the number of through holes 25 a. Furthermore, the amountand concentration of the liquid added dropwise to the wells 26 areadjusted such that approximately single DNA molecule is put into eachwell 26. For example, in the present embodiment, the amount of liquidthat fills the wells 26 is 0.5 μL for the entirety of the reactioncontainer, and 0.5 μL of the liquid is distributed into the plurality ofwells 26.

Thereafter, as shown in FIG. 8, from the inlet portion, the oil sealingsolution 22 is fed into the flow channel 31 formed by the base portion23 and the cover portion 27. In a state where the reagent has beendistributed into the buffer, the oil sealing solution 22 seals theliquid in the plurality of wells 26, and as a result, the plurality ofwell 26 becomes a plurality of independent reaction chambers 36(reaction containers for nucleic acid detection). That is, in thepresent embodiment, because the oil sealing solution 22 covers each ofthe wells 26, each of the wells 26 becomes independent from each othersimilarly to the microspaces disclosed in the first embodiment.Furthermore, in the gap between the base portion 23 and the coverportion 27, the oil sealing solution 22 pushes the liquid on the outsideof the plurality of wells 26 out of the outlet portion (Step S203 shownin FIG. 9).

Then, the array device 20 in which each of the wells 26 is filled withthe INVADER reaction reagent and DNA is incubated in an oven at 62° C.,for example (Step S204 shown in FIG. 9). By the incubation, signalamplification performed in an isothermal INVADER reaction suitablyproceeds.

Subsequently, the array device 20 in which each of the wells 26 isfilled with the INVADER reaction reagent and DNA is taken out after apreset time, and the number of wells giving off fluorescence and theamount of fluorescence are measured (Step S205 shown in FIG. 9).

That is, the biomolecule analysis method using the biomolecule analysiskit 100A according to the present embodiment includes a step (reagentfeeding step) of feeding a reagent into a flow channel in a reactioncontainer having the flow channel and a plurality of container-shapedportions such that the plurality of wells is filled with the reagent,and a step (sealing step) of sealing the reagent in the plurality ofwells with an oil sealing solution by feeding the oil sealing solutioninto the flow channel after the reagent feeding step such that theplurality of wells becomes a plurality of independent reactioncontainers for nucleic acid detection.

In the present embodiment, a detection system may also be adopted whichdetects, in addition to fluorescence, emission of visible light, colordevelopment, a change in pH, a change in electric potential, or the likeas a signal. In addition, the constitution of the present embodiment canbe adopted for analyzing proteins.

In the present embodiment, each reagent contains the adsorptioninhibitor, and accordingly, the constituents of the reagent can beprevented from being adsorbed onto the inner surface of the reactioncontainer 30 of the biomolecule analysis kit 100A. The adsorptioninhibitor may be contained in all of the reagents or some of thereagents.

The reagents may contain a substance reducing the surface tension of theconstituents of the reagents instead of the adsorption inhibitor. Forexample, a surfactant reduces the surface tension of the reagents.Accordingly, in order to fill each of the wells with the reagents, it iseffective for the reagents to contain a surfactant.

EXAMPLES First Example

Next, an example demonstrated for checking the operation and effect ofthe biomolecule analysis method according to the first embodiment of thepresent invention will be described. FIG. 3 shows fluorescence imagesshowing the results of a fluorescence amount measurement test in thepresent example. FIG. 4 is a graph showing the results of a fluorescenceintensity measurement test in the present example. In FIG. 4, theabscissa shows the reaction time, and the ordinate shows thefluorescence intensity. FIG. 5 is a table showing the results of areaction time measurement test in the present example. In FIG. 5,“Excellent” means that the quantification properties are excellent, and“Poor” means that the quantification properties are poor in the presentexample.

<Test of Counting Number of Wells Giving Off Fluorescence>

First, the microspaces 11 of the reaction container 10 were filled withthe INVADER reaction reagent and 3 kinds of artificial synthetic DNA. Atthis time, the concentration of the artificial synthetic DNA was set tobe 30 pM at which a single molecule was put into a single well, 50 nM atwhich 1,666 molecules were put into a single well, and 0 M at which nomolecule was put into a single well.

Then, the reaction container 10 was incubated in an oven at 62° C., andafter 0 minute (0 min), 10 minutes (10 min), and 15 minutes (15 min),the condition of the reaction container 10 was checked.

As shown in FIG. 3, if the DNA concentration is equal to or greater than30 pM, the fluorescence amount varies in almost all of the microspaces11 with respect to the background observed in a case where the DNAconcentration is 0 M, and it is understood that DNA is present.

<Fluorescence Intensity Measurement Test>

Next, the reaction container 10 filled with the artificial synthetic DNAset as above was incubated in an oven at 62° C. Furthermore, in order tocheck the condition of the reaction container after 0 minutes, 10minutes, and 15 minutes, images of 5 wells were selected at each DNAconcentration, and the average of the fluorescence amount of 21 pixelsin each image was determined. Herein, the wells after the reaction weremeasured using a fluorescence microscope (ZEISS, AX10), an object lens(EC Plan-Neofluar 40× oil NA 1.3), a light source (LEJ, FluoArc00 1.26AUsable with HBO 10), a sensor (Hamamatsu Photonics K.K., EM-CCD C9100),a filter (OLYMPUS CORPORATION, U-MNIBA2), and analysis software(Hamamatsu Photonics K.K., AQUACOSMOS 2.6: exposure time 64.3 ms, EMgain 180, offset 0, binning X1).

As shown in FIG. 4, when the DNA concentration was 30 pM at which asingle molecule was accommodated in a single microspace 11, thefluorescence with intensity distinguishable from the case where the DNAconcentration was 0 M was detected.

<Reaction Time Measurement Test>

Thereafter, the reaction time in the analysis method of the related artwas compared with the reaction time in the analysis method of thepresent invention. For the reaction time measurement test, as methodscompared with the present invention, a method of performing a digitalPCR reaction using a reagent in an amount of 1 nanoliter (nl)×multiplewells (Comparative example 1), a method of performing a PCR reactionusing a reagent in an amount of 20 microliters (μl) (Comparative example2), a method of performing PCR+INVADER reaction using a reagent in anamount of 20 μl (Comparative example 3), a method of performing anINVADER reaction using a reagent in an amount of 20 μl (Comparativeexample 4), and a method of performing a digital ELISA reaction using areagent in an amount of 100 femtoliters (fl)×multiple wells (Comparativeexample 5) were adopted.

As shown in FIG. 5, the reaction time measurement test revealed that inComparative example 1 in which a digital PCR reaction was performedusing a reagent in an amount of 1 nl×multiple wells, 60 minutes wereconsumed as the reaction time, the temperature condition wasnonisothermal, and the quantification properties were excellent. InComparative example 2 in which a PCR reaction was performed using areagent in an amount of 20 μl, 60 minutes were consumed as the reactiontime, the temperature condition was nonisothermal, and thequantification properties were not excellent. In Comparative example 3in which PCR+INVADER reaction were performed using a reagent in anamount of 20 μl, 60 minutes were consumed as the reaction time, thetemperature condition was nonisothermal, and the quantificationproperties were not excellent.

In Comparative example 4 in which an INVADER reaction was performedusing a reagent in an amount of 20 μl, 120 minutes were consumed as thereaction time, the temperature condition was isothermal, and thequalification properties were not excellent. In Comparative example 5 inwhich a digital ELISA reaction was performed using a reagent in anamount of 100 fl×multiple wells, 15 minutes were consumed as thereaction time, the temperature condition was isothermal, and thequantification properties were excellent.

In contrast, in the present example in which a digital INVADER reactionwas performed using a reagent in an amount of 100 fl×multiple wells,only 10 minutes were consumed as the reaction time, the temperaturecondition was isothermal, and the quantification properties wereexcellent. Therefore, it has become evident that because a digitalINVADER reaction was performed using a reagent in an amount of 100fl×multiple wells in the present example, the above excellent resultswere obtained.

Second Example

Next, an example demonstrated for checking the operation and effect ofthe biomolecule analysis method according to the second embodiment ofthe present invention will be described. FIG. 10 is a fluorescence imageshowing wells in the present example. FIGS. 11A to 11M are fluorescenceimages showing the results of a fluorescence amount measurement test inthe present example. FIG. 12 is a graph showing the results of afluorescence intensity measurement test in the present example. In FIG.12, the abscissa shows the concentration of TWEEN 20, and the ordinateshows the fluorescence intensity.

<Preparation of Array Device for Nucleic Acid Quantification>

A glass substrate having a thickness of 0.5 mm was spin-coated withCYTOP (registered trademark) (manufactured by ASAHI GLASS CO., LTD.) andthen baked for 1 hour at 180° C. The thickness of the formed CYTOP was 3μm. After being spin-coated with CYTOP, the substrate was coated with apositive photoresist, and a pattern was formed thereon by using aphotomask. Then, by using 02 plasma, CYTOP was dry-etched. In order toremove the residual photoresist on the surface, the surface was washedand rinsed with acetone and ethanol.

As shown in FIG. 10, each of the wells (microspaces) formed of CYTOP hada diameter of 5 μm and had a volume that makes it possible to detect asignal within several minutes by the INVADER reaction. In a single baseportion, a well array consisting of 100 blocks was provided, and each ofthe blocks had 10,000 wells. Therefore, a total of 1,000,000 wells wereformed. As shown in FIG. 6, a glass plate having a feeding port (inletportion: not shown in the drawing) was bonded to the base portion byusing a double-sided tape which had a thickness of 50 μm and wasprocessed to have a flow channel shape.

<Feeding Mixed Liquid of Sample and Detection Reaction Reagent>

The ease of forming liquid droplets by the concentration of TWEEN 20 asa surfactant was checked.

First, through the feeding port, a wash buffer containing the surfactantwas fed into the array device for nucleic acid quantification. Then, 22μl of an INVADER reaction reagent (detection reaction reagent 21: 1 μMallele probe, 1 μM INVADER oligo, 1 μM FAM-labeled arm, 10 mM MOPS pH7.5, 6.25 mM MgCl₂, 50 U/μL cleavase, TWEEN 20) and DNA which was asubstance as a target of analysis were fed into the array device fornucleic acid quantification.

Thereafter, through the feeding port, 80 μl of FC40 (oleaginous sealingsolution 22) as a fluorine-based liquid was fed into the array devicesuch that the reagent was distributed into and filled the respectivewells. By heating the array device on a hot plate at 63° C., the INVADERreaction was performed.

Subsequently, by using a fluorescence microscope (manufactured byOLYMPUS CORPORATION), at points in time when 10 minutes and 20 minuteselapsed at 63° C., the fluorescence from each well was detected. Theexposure time was set to be 100 msec for a bright field, 2,000 msec forNIBA, and 2,000 msec for mCherry.

FIGS. 11A to 11G show the results obtained by observing each well with amicroscope after 10 minutes of heating. FIGS. 11H to 11M shows theresults obtained by observing each well with a microscope after 20minutes of heating.

When the concentration of TWEEN 20 was 0%, fluorescence was alsodetected from the region positioned between the adjacent wells, andaccordingly, digital measurement could not be accurately performed.Presumably, this is because the droplets of the reaction solution in theadjacent wells were bonded to each other, and thus the sample reacted inthe region positioned between the adjacent wells. As another reason, itis considered that although the droplets of the reaction solution in theadjacent wells were not bonded to each other, the residual sample on thesurface of the base portion positioned between the adjacent wellsreacted. In contrast, it was confirmed that if the concentration ofTWEEN 20 contained is equal to or greater than 0.0005%, the droplets ofthe reaction solution are separated from each other.

Furthermore, it was confirmed that if the concentration of TWEEN 20contained is equal to or greater than 0.001% in the wells havingundergone 20 minutes of heating, the droplets of the reaction solutionare separated from each other. That is, it is considered that in a casewhere heating is performed for a long period of time, if theconcentration of TWEEN 20 contained is equal to or greater than 0.001%,the reproducibility is further improved compared to a case where heatingis performed for a short period of time.

FIG. 12 is a graph showing the values of fluorescence intensityassociated with the concentration of TWEEN 20. As the concentration ofTWEEN 20 increased, the fluorescence intensity weakened. That is, abehavior in which the increase in the concentration of TWEEN 20 hinderedthe reaction was confirmed. Therefore, an optimal concentration of TWEEN20 is assumed to be less than about 5%. In addition, it is consideredthat, from the viewpoint of costs, the concentration of TWEEN 20 is morepreferably equal to or less than 0.5%.

10 μl of the same reagent was dispensed into a 96-well plate, and thereactivity at a volume of 10 μl was detected using LightCycler LC480(manufactured by Roche Life Science). The temperature condition of theLightCycler was kept constant at 63° C. With the LightCycler, a reactionwas performed in the same composition as described above. As a result,it was confirmed that the increase of the fluorescence signal of theINVADER reaction was constant regardless of the concentration of TWEEN20. Therefore, it was understood that the surfactant contributes not tothe improvement of the reactivity of the enzyme but to the stability ofthe liquid droplets.

The surfactant should be added at such a concentration that can preventthe substance as a target of detection contained in the reagent frombeing adsorbed onto CYTOP, glass, or the like. In a case where othersurfactants such as TRITON-X100 are used, the optimal concentration maybe changed, but the case of TWEEN 20 can be taken into consideration.

Third Example

Next, an example demonstrated for checking the operation and effect ofthe biomolecule analysis method according to the second embodiment ofthe present invention will be described. FIG. 13 is a view showing theoperation and effect of the biomolecule analysis method according to thesecond embodiment of the present invention. FIGS. 14A to 14F arefluorescence images showing the results of a fluorescence amountmeasurement test in the present example. In FIG. 13, “Excellent” in thecolumn of reactivity means that the reactivity is excellent, and “O” inthe column of liquid droplet means that fluorescence was not observed inthe region between two adjacent wells. Furthermore, in FIG. 13, “A” inthe column of liquid droplet means that, although fluorescence wasobserved in the region between two adjacent wells, the measurement ofconcentration was not affected by the fluorescence. In addition, in FIG.13, “X” in the column of liquid droplet means that fluorescence wasobserved in the region between two adjacent wells, and thus the digitalmeasurement could not be accurately performed in some cases when theregion between the two adjacent wells is used.

In the present example, by using the INVADER reaction reagent and DNAused in the second example, as shown in FIG. 13, a reaction wasperformed by changing the conditions such as whether or not the wellwill be washed, whether or not a surfactant will be added to the washbuffer, and whether or not a surfactant will be added to the reactionreagent, and from the obtained fluorescence image, the state andreactivity of the liquid droplets were checked. As a surfactant, 0.05%TWEEN 20 was added to the wash buffer or the reaction reagent. Otherconditions were the same as in the second example.

In samples 1 and 2, the wells were washed by adding the surfactant tothe wash buffer. In samples 3 and 4, the wells were washed withoutadding the surfactant to the wash buffer. In samples 5 and 6, the wellswere not washed. Furthermore, the surfactant was added to the reactionreagent for the samples 1, 3, and 5. In contrast, the surfactant was notadded to the reaction reagent for the samples 2, 4, and 6. Thereactivity was excellent in all of the samples. In addition, as isevident from the sample 2, it was understood that, even in a case wherethe surfactant is not added to the reaction reagent, as long as the washbuffer contains the surfactant, liquid droplets are excellently formed,and the reactivity becomes excellent.

As described above, with the biomolecule analysis method and thebiomolecule analysis kit 100 according to the first embodiment of thepresent invention and with the biomolecule analysis method and thebiomolecule analysis kit 100A according to the second embodiment of thepresent invention, it is possible to rapidly and quantitatively analyzebiomolecules by performing an enzymatic reaction in the microspaces 11or the wells 26.

In the biomolecule analysis method and the biomolecule analysis kit 100according to the first embodiment of the present invention and with thebiomolecule analysis method and the biomolecule analysis kit 100Aaccording to the second embodiment of the present invention, an INVADERmethod is used as an enzymatic reaction. As a result, PCR amplificationis not required, and an isothermal reaction can be performed. Therefore,the device constitution and the analysis procedure can be simplified.

In the biomolecule analysis method and the biomolecule analysis kit 100according to the first embodiment of the present invention and in thebiomolecule analysis method and the biomolecule analysis kit 100Aaccording to the second embodiment of the present invention, a reactionis performed in the microspaces 11 and the wells 26 each of which has avolume to accommodate a single molecule as a target of analysis.Therefore, the time taken until the signal is saturated can beshortened.

In the biomolecule analysis method and the biomolecule analysis kit 100according to the first embodiment of the present invention and in thebiomolecule analysis method and the biomolecule analysis kit 100Aaccording to the second embodiment of the present invention, thereaction time is shorter and the SN ratio is higher, compared to therelated art.

In the biomolecule analysis method and the biomolecule analysis kit 100according to the first embodiment of the present invention and in thebiomolecule analysis method and the biomolecule analysis kit 100Aaccording to the second embodiment of the present invention, theenzymatic reaction is an isothermal reaction. Therefore, the enzymaticreaction that is more stable compared to a nonisothermal reaction can becarried out, and the reproducibility is high.

In the biomolecule analysis method and the biomolecule analysis kit 100according to the first embodiment of the present invention and in thebiomolecule analysis method and the biomolecule analysis kit 100Aaccording to the second embodiment of the present invention, theenzymatic reaction is an INVADER reaction. Therefore, the time taken forsignal detection/determination can be further shortened compare to theprocess requiring PCR.

In the biomolecule analysis method and the biomolecule analysis kit 100according to the first embodiment of the present invention and in thebiomolecule analysis method and the biomolecule analysis kit 100Aaccording to the second embodiment of the present invention, each of themicrospaces 11 or the wells 26 has a volume of equal to or less than 100picoliters. Therefore, the amount of the reagent consumed for analysiscan be reduced.

In the above embodiments, examples in which the low-adsorptionstructural portion and the adsorption inhibitor are used in combinationare described. However, as long as at least one of the low-adsorptionstructural portion and the adsorption inhibitor is adopted, it ispossible to more rapidly and quantitatively analyze biomoleculescompared to a case where none of the low-adsorption structural portionand the adsorption inhibitor is adopted.

What is claimed is:
 1. A biomolecule analysis kit comprising: an arraydevice including: a base portion being a flat plate, a reactioncontainer configured to perform an enzymatic reaction, the reactioncontainer including a plurality of wells to which sample biomolecules astargets of biomolecule analysis is to be supplied, the plurality ofwells being formed in the base portion, a flat cover portion superposedon the base portion and the plurality of wells, a flow channel formedbetween the flat cover portion and the plurality of wells and connectingthe plurality of wells through a gap between the base portion and theflat cover portion, the flow channel having: an inlet portion, and anoutlet portion, wherein at least one well among the plurality of wellsis positioned between the inlet portion and the outlet portion; and areagent configured to be supplied to the plurality of wells and to beused for the enzymatic reaction which generates a signal, wherein thereagent contains an adsorption inhibitor to reduce an adsorption rate ofat least one of the sample biomolecules or the reagent with respect tothe base portion; and the base portion has a hydrophobic surface at aregion positioned between wells that are adjacent, among the pluralityof wells.
 2. The biomolecule analysis kit according to claim 1, whereinthe adsorption inhibitor is a surfactant.
 3. The biomolecule analysiskit according to claim 2, wherein the surfactant is a nonionicsurfactant.
 4. The biomolecule analysis kit according to claim 3,wherein the nonionic surfactant is polyethylene glycol sorbitanmonolaurate.
 5. The biomolecule analysis kit according to claim 3,wherein the nonionic surfactant is4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol.
 6. Thebiomolecule analysis kit according to claim 2, wherein the concentrationof the surfactant is equal to or greater than 0.0005 vol % and equal toor less than 5 vol %.
 7. The biomolecule analysis kit according to claim1, further comprising: an oil sealing solution configured to seal eachof the plurality of wells filled with the reagent.
 8. The biomoleculeanalysis kit according to claim 7, wherein the oil sealing solution isconfigured to cover each of the plurality of wells and the hydrophobicsurface.
 9. The biomolecule analysis kit according to claim 1, whereinthe volume of each well of the wells is equal to or less than 100picoliters.
 10. The biomolecule analysis kit according to claim 1,further comprising: a wash buffer to be fed into the gap between thebase portion and the flat cover portion.
 11. The biomolecule analysiskit according to claim 10, wherein the wash buffer contains anadsorption inhibitor.
 12. The biomolecule analysis kit according toclaim 10, wherein the wash buffer is intermixable with the reagent. 13.The biomolecule analysis kit according to claim 1, further comprising: awash buffer filled in the gap between the base portion and the flatcover portion.
 14. The biomolecule analysis kit according to claim 13,wherein the wash buffer contains an adsorption inhibitor.
 15. Thebiomolecule analysis kit according to claim 13, wherein the wash bufferis intermixable with the reagent.
 16. The biomolecule analysis kitaccording to claim 1, wherein the base portion is transparent.